Transcriptional regulation of c-fos and immediate early genes

描述（由申请人提供）：本提案的目的是表征生长因子受体用于激活即刻早期基因的信号通路。原型即刻早期基因c-fos受血清应答元件（SRE）调节，其结合血清应答因子（SRF）。SRF由两条途径激活。一个是SRF辅因子的MARK磷酸化，三元复合物因子（TCF），第二个是通过小GTdR RhoA的信号传导。在这个提议中，RhoA激活c-fos启动子的途径将被研究。将研究SRF辅激活因子MKL 1/2，因为它们已被证明是血清和RhoA激活SRF靶基因所必需的。MKL 1响应于细胞的血清处理而迅速磷酸化。诱导磷酸化的位点已被绘制，并将测试其对靶基因血清诱导的要求。然后将鉴定负责的蛋白激酶。还将确定MKL 1结构域在其调节中的作用，并将分离与关键结构域复合的蛋白质。已提出RhoA信号传导至SRF涉及肌动蛋白丝的变化。然而，RhoA的突变反对这种模型。这表明一种新的RhoA效应子被用于向SRF发出信号。参与SRF活化的RhoA的靶标将使用缺乏与RhoA突变体的结合作为选择标准来纯化。我们通过微阵列分析鉴定了许多血清诱导基因，并发现一个子集依赖于MKL 1的诱导。目前还不清楚有多少其他基因被TCF甚至SRF激活。我们将开发这些因子缺陷的细胞系，并使用微阵列来确定哪些机制调节整个类别的立即早期基因。这可能会识别出独立于这些因素激活的基因。这些基因将被映射为新的调节因子，以确定新的信号通路。我们还在芯片中发现了许多血清阻遏基因。这种抑制的机制尚未被广泛研究，许多被抑制的基因具有抗增殖作用。我们将描述这些基因的调控，以确定新的信号通路在立即早期反应。

DESCRIPTION (provided by applicant): The aim of this proposal is to characterize the signaling pathways utilized by growth factor receptors to activate immediate early genes. The proto-type immediate early gene c-fos is regulated by a Serum Response Element (SRE), which binds Serum Response Factor (SRF). SRF is activated by two pathways. One is MARK phosphorylation of the SRF cofactors, the ternary complex factors (TCFs), and the second is signaling through the small GTPase RhoA. In this proposal the pathways used by RhoA to activate the c-fos promoter will be studied. The SRF coactivators MKL1/2 will be studied as they have been shown to be required for serum and RhoA activation of SRF target genes. MKL1 is rapidly phosphorylated in response to serum treatment of cells. The sites of inducible phosphorylation have been mapped and will be tested for their requirement for serum induction of target genes. The responsible protein kinase will then be identified. The role of domains of MKL1 in its regulation will also be determined and complexing proteins to critical domains will be isolated. RhoA signaling to SRF has been proposed to involve changes in actin filaments. However, mutations in RhoA argue against this model. This suggests that a novel RhoA effector is used to signal to SRF. Targets of RhoA involved in SRF activation will be purified using lack of binding to RhoA mutants as selection criteria. We have identified many serum inducible genes by microarray analysis and found that a subset is dependent upon MKL1 for its induction. It is unclear how many of the other genes are activated by TCF or even by SRF. We will develop cell lines defective in these factors and use microarrays to determine which mechanisms regulate the entire class of immediate early genes. This will likely identify genes activated independently of these factors. These genes will be mapped for novel regulatory factors to identify new signaling pathways. We also found in microarrays that there are many serum repressible genes. The mechanisms for this repression have not been extensively studied and many of the repressed genes have anti-proliferative effects. We will characterize regulation of these genes to identify new signaling pathways in the immediate early response.